Fast acting inhibitor of gastric acid secretion

ABSTRACT

The present invention relates to the use of pharmaceutically acceptable zinc salts, preferably water soluble zinc salts alone or optionally, in combination with one or more of a protein pump inhibitor (PPI), H2 blocker, anti- H. pylori  antibiotic/antimicrobial, cytoprotective agent or a combination agent as otherwise described herein for providing fast action with optional long duration effect in reducing gastric acid secretion, raising the pH of the stomach during resting phase as well as decreasing the duration of stomach acid release during a secretagogue phase and for treating conditions including gastroesophageal reflux disease (GERD), non-erosive reflux disease (NERD), Zollinger-Ellison syndrome (ZE disease), ulcer disease, and gastric cancer, as well as preventing or reducing the likelihood of ulcer disease. In addition, the present methods are useful for treating patients who are non-responsive to proton pump inhibitors (PPI) and as an alternative to traditional therapies or conditions which are caused by rapid and complete inhibition of secretagogue induced acid secretion.

RELATED APPLICATIONS

This application claims the benefit of priority of U.S. provisionalapplications No. US60/762,595, filed Jan. 27, 2006, US60/764,834, filedFeb. 3, 2006 and US60/850,891, filed Oct. 11, 2006, each of saidapplications being incorporated by reference herein in their entirety.

GOVERNMENT RIGHTS STATEMENT

This invention was made with government support under DK050230 awardedby National Institutes of Health. The government has certain rights inthe invention.

FIELD OF THE INVENTION

The present invention relates to the use of pharmaceutically acceptablezinc salts, preferably water soluble zinc salts alone or optionally, incombination with one or more of a protein pump inhibitor (PPI), H2blocker, anti-H. pylori antibiotic/antimicrobial, cytoprotective agentor a combination agent as otherwise described herein for providing fastaction with optional long duration effect in reducing gastric acidsecretion, including acid secretion in the fundus (by inhibitingvacuolar H⁺-ATPase) and upper body region of the stomach (by inhibitingH⁺/K⁺-ATPase), thus raising the pH of the stomach during resting phaseas well as decreasing the duration of stomach acid release during asecretagogue phase and for treating conditions includinggastroesophogeal reflux disease (GERD), non-erosive reflux disease(NERD), Zollinger-Ellison syndrome (ZE disease), ulcer disease, andgastric cancer, as well as preventing or reducing the likelihood ofulcer disease. In addition, the present methods are useful for treatingpatients who are non-responsive to proton pump inhibitors (PPI) and asan alternative to traditional therapies or conditions which are causedby rapid and complete inhibition of secretagogue induced acid secretion.

BACKGROUND OF THE INVENTION

The generation of concentrated 0.16N hydrochloric acid by the mammalianparietal cell involves a complex combination of neuronal and hormonalregulatory feedback loops¹⁻³ Following activation of the cell there is acomplex cellular transfer of ions that allows for the formation ofacid⁴⁻⁷. A disruption in any of these components (secretory receptors,or ion transporters) can lead to either a cessation in the secretion ofacid, or in the hypersecretion of acid. In the latter over 30 millionpatients per year suffer from symptoms of acid related diseases with thenumbers increasing yearly⁸⁻¹¹. Clinically the uncontrolled release orthe continued hypersecretion of acid can lead to changes in both gastricand intestinal epithelium, but can in more serious cases lead toerosions of the esophagus that can result in metaplasia and death¹²⁻¹⁴.Recent evidence has also emerged that prolonged recurrent periods ofhypersecretory states can lead to gastric carcinoid formation¹⁵.

In an attempt to design therapies to prevent hyperacid secretion avariety of approaches have been employed in recent years with two of themost successful being: a) inhibition of the Histamine receptor on thebasolateral membrane of the parietal cell, b) proton pump specific drugstargeted against the H,K-ATPase (the so called proton pump inhibitors;PPI)¹⁶⁻¹⁸. Both of these therapies have greatly improved the quality oflife for patients suffering from this disease, however there is an everincreasing number of patients that have experienced recurrent diseasewhile still taking the drugs^(19, 20). Despite their high degree ofefficacy and worldwide clinical use, failure in the treatment of acidrelated diseases has been reported and the degree and speed of onset ofsymptom relief are important to patients²¹. It has been estimated thatabout 30% of GERD patients remain symptomatic on standard dose of PPI²².Furthermore PPI's have a short plasma half life which often leads tonocturnal acid breakthrough²³. Therapeutic oral doses of PPIs reachsteady state and thus achieve their maximal effective levels after 4-5days with typical dosing regimens²⁴. This slow and cumulative onset ofeffect of PPIs relates to their ability to inhibit only those pumpswhich are active when the PPI drug is available. After PPIadministration, there is a return of acid secretion that is partly dueto de novo synthesis of the enzyme²⁵.

Zinc is an essential part of the diet that all cells require in order tomaintain membrane integrity and function. Deficiency in intracellularzinc leads to apoptotic events, and cell death²⁶⁻³⁰. Previous studieshave investigated the potential role of zinc in the proliferation andgeneration of the protective barrier, namely the mucous gel layer at thesurface of the stomach³¹⁻³⁴. These studies falsely attributed thereduction in acid secretion to an increase in the thickness of the gellayer³³⁻³⁵.

Gastric acid aids protein digestion; facilitates the absorption of iron,calcium, and vitamin B12; and prevents bacterial overgrowth. When levelsof acid and proteolytic enzymes overwhelm the mucosal defensemechanisms, ulcers occur. To avoid damage that is associated with theseharsh conditions, gastric acid must be finely regulated by overlappingneural (e.g. acetylcholine), hormonal (e.g. gastrin and ghrelin), andparacrine (e.g. histamine and somatostatin) pathways, and more recentlyvia the Calcium Sensing Receptor. Any long term alterations in any ofthese regulatory pathways leads to cell and tissue destruction andclinical manifestations such as peptic ulcer diseases, orgastroesophageal reflux disease (GERD). Two methods are commonlyemployed to treat the overproduction of acid: a) surgically, byelimination of the neuronal element (vagotomy) or b) pharmacologically,either through histamine 2 receptor antagonists or proton pumpinhibitors (PPI's) or a combination of both.

PPI's such as omeprazole are irreversible inhibitors of the gastricH+/K+-ATPase, recently various derivatives of the parent compoundomeprazole that bind to multiple cysteine residues on the exofacialsurface of the H+/K+-ATPase have been developed in hopes of having atighter molecular binding, and longer action have been employed. Bothrabeprazole, and lansoprazole are examples of these multiple bindingdrugs and are activated in the acidic lumen of the gastric gland andmodify the cysteine residues located on the luminal surface of theH+/K+-ATPase. In the resting cell the acid secreting pumps areinternalized in a system of tubular vesicles, and are in such aconformational state that the PPIs can only inhibit the H+/K+-ATPaseswhich have already been activated and transferred to the apical surfaceof the parietal cell.

Although optimizing pharmacological profiles within the PPI class mayprovide some clinical benefit, other areas of research may prove to bemore fruitful and furthermore the fine tuning of the acid secretoryprocess is still not completely understood and remains an importanttarget for therapies to modulate gastric acid secretion.

Zinc is required for a large number of biological processes includinggene expression, replication, membrane stability, hormonal storage andrelease and as a catalytic component for enzymes. There has been noinvestigation of the actions of zinc at the cellular level relating toeffects on acid secretion.

Helicobacter pylorus resides within the mucous layer of the humangastric mucosa. Due to extremely low pH, the stomach is a hostileenvironment to most other microorganisms. The ability of H. pylori toflourish in the stomach has been attributed to protective mechanismssuch as its production of urease, protecting the bacterium from gastricacidity by creating a basic microenvironment, See, Taylor and Blaser,Epidemiol Rev, 13:42-59, (1991).

The stomach is a large organ that can be divided into 3 main zones thatare involved in the process of digestion of foodstuff and thesterilization of liquids and water. When defining the functional processof the stomach it has been commonly divided into two zones: UpperStomach, and Lower Stomach. The upper stomach, is thought to be composedof the fundus and upper body, and shows low frequency, sustainedcontractions that are responsible for generating a basal pressure withinthe stomach. Of note is that these tonic contractions also generate apressure gradient from the stomach to small intestine and areresponsible for gastric emptying. Interestingly, when swallowing foodand the consequent gastric distention that occurs acts to inhibitscontraction of this region of the stomach, allowing it to balloon outforming a large reservoir without a significant increase in pressure.The lower stomach is thought to be involved in the grinding andliquefaction of the foodstuffs by the secretion of HCl from the parietalcells found in this section of the stomach.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-E shows the original tracing of basal acid secretion, histamineinduced acid secretion and inhibition by ZnCl₂. Single human and ratgastric glands were isolated, loaded with the pH-sensitive dye BCECF tomeasure intracellular pH over single parietal cells and the pH_(i)recovery rate was calculated from the slope after an acid load using theNH₄Cl prepulse technique. (A,C) Intracellular alkalinization stimulatedby histamine (100 μM) in the absence of extracellular Na⁺ as a functionof H⁺/K⁺-ATPase in gastric glands. (B,D) Histamine induced proton effluxfrom gastric glands can be blocked by 300 umol ZnCl₂. (E) Bar graphsummarizing data as means SE (control: n=32 cells, 3 gland, 3 animals;histamine: n=120 cells, 15 glands, 8 animals; histamine+ZnCl₂: n=60cells, 6 gland, 4 animals).

FIG. 2 shows that ZnCl₂ inhibits acid secretion in a dose dependentmanner. ZnCl₂ concentration dependence of H⁺/K⁺-ATPase activity(intracellular alkalinization expressed as ΔpH/min) in the presence of100 μmol histamine in comparison to basal and histamine induced acidsecretion. (n=40 cells, 3-4 glands, 3-4 animals for each ZnCl₂concentration).

FIG. 3 show the fast onset inhibitory effect and reversibility withZnCl₂. (A) original tracing shows the fast inhibitory effect of ZnCl₂ onhistamine induced acid secretion. Histamine (100 μM) was added throughthe whole experiment. When the intracellular alkalinization(protonefflux) was observed, ZnCl₂ (300 μM) was added to the superfusionbath. The acid secretion was abolished after a few seconds (flat middlepart). After the removal of ZnCl₂ out of the perfusion bath the drug waswashed out and the increase of the intracellular pH continued. (B)Original tracing shows the reversibility after the cells where incubatedand superfused over 20 min with ZnCl₂ (300 μM) and histamine (100 μM).After removal of ZnCl₂ out of the superfusion bath the intracellularalkalinization (proton extrusion) occur.

FIG. 4 shows acid secretion after oral ZnCl₂ application. 300 μmol ZnCl₂was added to the drinking water. Animals ate and drank as much ascontrol animals. Prior the experiment they were fasted for 12-18 hours.The histamine induced acid secretion was measured as described before.The cells of the ZnCl₂ treated animals showed a lower rate of protonefflux. 150 mg/kg/d: 0.022±0.0045; (n=60 cells, 10 glands, 3 animals),0.05 mg/kg/d: 0.034±0.0036; (n=60 cells, 6 glands, 4 animals).

FIG. 5 shows that ZnCl₂ inhibits gastric acid secretion in freshlyisolated rat whole stomach preparation. Ex vivo rat whole stomachpreparations were incubated in HEPES-buffered Ringer solution (control:n=9), HEPES-buffered Ringer solution plus 100 μM histamine (n=8), orHEPES-buffered Ringer solution plus 100 μmol histamine and 300 μmolZnCl₂ (n=8). Stomach preparations incubated with histamine and ZnCl2 hada higher pH than those in HEPES-buffered Ringer solution and histamineand their pH was similar to the pH of the control stomach.

FIG. 6 shows measurements of whole stomach intraluminal pH using anumber of zinc salts according to the present invention. Isolated wholestomach preparations from rats were cannulated at the esophageal andduodenal junction and perfused in vitro with 37° C. pH 7.4 Ringerssolution. The blood perfusate was then exposed to 100 μM Histamine toinduce acid secretion. The lumen of the stomach was infused with 0.5 ccof non-buffered isotonic saline. In some studies one of the followingzinc salts was added to the lumen perfusate at a final concentration of300 μM (zinc chloride, zinc sulfate, zinc acetate, zinc citrate). Thedata are the sum of 5 separate stomachs from 5 separate animals for eachof the columns. Data are the mean of all studies with the standard errorof the mean displayed.

FIGS. 7A-C show the immunohistochemistry in rat stomach fundus. (A)Immunolocalization of the gastric H⁺, K⁺-ATPase α subunit in rat fundicgland parietal cells (40×). (B) Fundic parietal cell Electron Microscopyof Gold Tagged H⁺, K⁺-ATPase protein. Here the nucleus, apical membraneand canaliculus like structure can be seen (8,000×). (C) Highermagnification (25,000×) of the same cell. Here the gold tagged H⁺,K⁺-ATPase protein can be seen distributed at the borders of thecanaliculus like structure (arrows). (In this figure: n=nucleus,c=canaliculus like structure, am=apical membrane)

FIGS. 8A-D show the original tracing of basal acid secretion andhistamine induced acid secretion in the gastric fundus and corpus.Single rat gastric glands were isolated, loaded with pH sensitive dyeBCECF to measure intracellular pH of single parietal cells and the pHirecovery rate was calculated from the slope after an acid load using theNH₄CL prepulse technique. (A) Original tracing of an F1 glandalkalinization (proton efflux) after removing Na⁺ out of the perfusionbath. (B) Intracellular alkalinization of an F1 gland stimulated byhistamine (100 μM) in the absence of extracellular Na⁺ as a function ofH⁺, K⁺-ATPase. (C) Tracing of a corpus gland alkalinization underresting condition. (D) Intracellular alkalinization of an corpus glandstimulated by histamine (100 μM) in the absence of extracellular Na⁺ asa function of H⁺, K⁺-ATPase.

FIG. 9 shows a secretagogue series of F1 glands F1 gland under basalcondition with no stimulation shows alkalinization rates of 0.039ΔpH_(i)/min±0.009 (n=52 cells/8 glands/5 animals). In the presence of100 μM histamine recovery rates were 0.042±0.007 ΔpH_(i)/min (n=64cells/8 glands/6 animals). In the presence of 100 μM acetylcholine F1glands alkalinized at a rate of 0.075±0.0015 ΔpH_(i)/min (n=86 cells/10glands/6 animals). In the presence of 100 μM pentagastrin F1 glands showalkalinization rates of 0.062±0.007 ΔpH_(i)/min (n=49 cells/6 glands/5animals).

FIGS. 10A-D shows original tracing of acid secretion comparing F1 glandsand Corpus glands with Omeprazole and AZD0865 Single rat gastric glandswere isolated, loaded with the pH sensitive dye BCECF to measureintracellular pH over single parietal cells and the pHi recovery ratewas calculated from the slope after an acid load using NH₄CL prepulsetechnique as described previously. (A) Original tracing of anintracellular pH measurement demonstrating a F1 gland alkalinizationafter stimulation by histamine (100 μM). This tracing shows thatomeprazole (200 μM) does not inhibit acid secretion in F1 glands. (B)Corpus gland tracing of intracellular alkalinization after stimulationwith histamine (100 μM). This tracing shows that Omeprazole (200 μmolar)inhibits acid secretion in the corpus with a intracellularalkalinization rate of (0.014±0.002 ΔpH_(i)/min. (C) Intracellulartracing of pH measurements demonstrating that AZD0865 does notcompletely inhibit proton extrusion in the fundus as it does in thecorpus. In fundic glands which have been exposed to 10 μM of AZD0865 theintracellular recovery is 0.031±0.006 ΔpH_(i)/min. (D) In the corpusAZD0865 shows strong inhibition of potassium dependant recovery withintracellular alkalinization rates of 0.021±0.008 ΔpH_(i)/min.

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to novel compositions and methods for therapid inhibition of acid secretion that has little to no potential forside effects. In a first aspect, the present invention relates to zinccompositions comprising at least one pharmaceutically compatible zincsalt (preferably a water soluble salt) in an effective amount whichproduces a rapid decrease (i.e., within a period of no greater thanabout 5 minutes, no greater than about 10 minutes, no greater than about20 minutes, no greater than about 30 minutes, no greater than one hour)of acid secretion in a patient's stomach with a resulting increase(elevation) in stomach pH to an intragastric pH level of at least about3.0-3.5, at least about 4.0, about 4.0 to about 5.0. In this aspect ofthe invention, a patient who is in need of an increase of stomach pH istreated with an effective amount of a pharmaceutically compatible zincsalt such that rapid onset of elevated pH within the stomach occurs.This method invention relies on the administration (preferably by, butnot limited to, ingestion) of an effective amount of at least onepharmaceutically compatible, preferably water-soluble zinc salt and inwhich a substantial portion dissolves in the gastric juices at low pH(generally less than about 2.0) and preferably within a range pH fromlow pH (about 1.0 to about 2.0) to higher pH (about 5.5 to about 7.5 orhigher) such that effective amounts of zinc salt may be administered toprovide an initial rapid inhibition of acid release and a subsequentmaintenance of inhibition of acid release in the stomach. In the presentinvention, inhibition of gastric acid is inhibited preferably within arapid period of about 20 minutes to about 1 hour (generally, within aperiod no greater than about 5 minutes, within a period no greater thanabout 10 minutes or within a period no greater than about 20 minutes,within a period no greater than about 30 minutes, within a period nogreater than about one hour).

The rapid decrease of acid secretion in the patient's stomach occursthroughout the stomach (in both the upper stomach and lower stomachthrough inhibition of H+/K+-ATPase), although localized effects ofcompounds according to the present invention in the upper stomach,especially in the fundic region of the stomach (through inhibition of asecond distinguishable protein H+-ATPase) and/or the upper body of theupper stomach (through inhibition of H+/K+-ATPase). Thus, an additionalaspect of the invention is directed to the use of effective amounts ofpharmaceutically acceptable zinc compounds for the inhibition ofH+/K+-ATPase (generally throughout the stomach, H+-ATPase (primarily inthe fundic region of the stomach) and preferably both. The finding thatthe present compounds may be used to inhibit H+-ATPase in the fundicregion has important clinical ramifications for the following reasons:

1) The erosion of the esophagus by exposure to acid has life threateningconsequences due to either internal bleeding, ulceration, and or gastriccarcinoid formation by the prolonged exposure to acid. Pursuant to thepresent invention, as is now demonstrated—glands in the fundus are indirect proximity to the esophageal juncture, that they will secrete acidand can be inhibited by compounds according to the present invention,thus making the present compounds particularly effective in treatingGERD, NERD and related conditions.2) There is an ever increasing number of patients that are becominginsensitive to PPI (proton pump inhibitors) and have recurrent symptomsof acid reflux disease. The protein that we identified in the fundicglands is not sensitive to PPI's and could be the reason that thesepatients do not respond to classical therapy.3) Patients on PPI's for long periods of time appear to show some“rebound” acid secretion. This result could again be linked to thefundic H⁺-ATPase, which we show is sensitive to Histamine and to thelevels of protons within the cell.

In preferred embodiments of this invention aspect, a single zinc saltwhich is water-soluble regardless of pH (i.e., within a range of pH fromabout 1.0 to about 7.5 or above) is preferred. Zinc chloride is thepreferred salt for use in the present invention. In alternativeembodiments, a mixture of a low pH soluble zinc salt with a high pHsoluble zinc salt or a zinc salt which may be readily absorbed throughthe small intestine (such as a zinc amino acid chelate compound),optionally in combination with a pharmaceutically acceptable buffer isprovided. In this aspect of the invention, an effective amount of a zincsalt selected from the group consisting of zinc chloride (ZnCl₂), zincacetate, zinc ascorbate, a zinc amino acid chelate (mono- orbis-chleate) and mixtures thereof, preferably a mixture of zinc chlorideand at least one of zinc acetate, zinc gluconate, zinc ascorbate, and azinc amino acid chelate is provided alone or in combination with apharmaceutically acceptable carrier, additive or excipient.

In various aspects, the present invention relates to the use of at leastone water-soluble zinc salt alone or in combination with at least onecompound/composition (within the context of the disease state orcondition to be treated) selected from the group consisting of atraditional proton pump inhibitor compound/composition, an H2 blocker,an antibiotic/antimicrobial agent (effective against H. pylori), acytoprotective agent or a mixture of these agents (Helidac, Prevpac) toprovide fast action in reducing gastric acid secretion, to lower the pHof the stomach, to prevent or reduce the likelihood of ulcer disease, totreat ulcer disease, to treat gastric cancer, to treat a disease orcondition selected from the group consisting of gastroesophageal refluxdisease (GERD), non-erosive reflux disease (NERD), Zollinger-Ellisonsyndrome (ZE disease), ulcer disease, and gastric cancer, as well aspreventing or reducing the likelihood of ulcer disease.

Pharmaceutical compositions comprising a mixture of zinc salts whichmaximize both immediate and extended release characteristics of thepresent invention, optionally in combination with a pharmaceuticallyacceptable carrier, additive or excipient and further optionally aneffective amount of additional agent selected from the group consistingof a proton pump inhibitor, an H₂ blocker, an anti-H. pyloriantibiotic/antimicrobial, a cytoprotective agent and a combination ofagents, are additional aspects of the present invention. Any one or moreof these compositions may be used within context to treat the variousconditions/disease states as otherwise disclosed herein.

DETAILED DESCRIPTION OF THE INVENTION

The following terms are used throughout the specification to describethe present invention.

The term “patient” or “subject” refers to an animal, preferably amammal, even more preferably a human, in need of treatment or therapy towhich compounds according to the present invention are administered inorder to treat a condition or disease state treatable using compoundsaccording to the present invention. Depending upon the disease orcondition treated the term patient refers to the animal treated for thatdisease within context.

The term “effective” is used to describe a treatment, compound,composition, component or a related aspect of the present invention,which, when used in context, produces an intended result which mayinclude the increase in pH in the stomach, the reduction of symptomsassociated with excess acid release or the favorable treatment of adisease state or condition. The term effective subsumes both an amountor concentration of one or more active agent(s) as described herein anda period of time which is consistent with producing an intended effect.

The term “pharmaceutically acceptable zinc salt” or zinc salt” used incontext, refers to a salt or salt combination which contains zinc,dissolves in the gastric juices at reduced pH and is absorbed to someextent in the gastric mucosa at a low pH of about 2 or less, at a higherpH of about 4.0 to 5.0 or above of the stomach and at the high pH's ofthe small intestine to reach and maintain effective concentrations ofzinc in the blood stream over a period of therapy. Exemplarypharmaceutically compatible zinc salts include both inorganic andorganic zinc salts, for example, zinc acetate, zinc ascorbate, zincbutyrate, zinc carbonate (soluble in dilute acid at low pH of thestomach), zinc citrate, zinc chloride, zinc iodide, zinc sulfate, zincgluconate, zinc glycerate, zinc glycolate, zinc formate, zinc lactate,zinc picolinate, zinc salicylate, zinc tartrate, zinc undenylate, andzinc amino acid chelates (including, depending on concentration, mono-and bis-chelates of L- or D-amino acids (preferably, the naturallyoccurring L-amino acid which may be more readily absorbed from thegastrointestinal tract) which complex or chelate with zinc includingpreferably, L-cysteine, L-cystine, L-N-acetylcysteine, L-histidine (alsoD-histidine), L-taurine, L-glycinate, L-aspartate and L-methionine,among others. Note that for purposes of the present invention, zincamino acid chelates are considered zinc salts. Preferably, thepharmaceutically acceptable zinc salt is water-soluble.

One of ordinary skill will recognize favorable zinc salts to use in thepresent invention. In aspects of the invention, at least onepharmaceutically compatible, water-soluble zinc salt is administered toa patient in order to provide a rapid inhibition of acid release in thestomach, resulting in an increase in stomach pH to above 4 (generallybetween about 4.0 and 5.0, in some cases above 5.0) for an extendedperiod of time, preferably at least 2 hours, 4 hours, 6 hours, 8 hours,10 hours, 12 hours, 16 hours, 20 hours or more. It is noted that incertain preferred aspects of the invention, the zinc salt or combinationof salts chosen to be administered to the patient may be adjusted toprovide an initial bolus concentration of zinc in the stomach at low pHin order to produce the rapid inhibition of acid release and rise in pHin the stomach to a level above about 4. In addition, a preferred zincsalt or salt combination inhibits acid release in the stomach at varyinglevels of acidity and pH—i.e., at a level which is quit acidic (pH, lessthan about 2.0) to a pH of about 4.0 or higher.

The term “providing fast action in reducing gastric acid secretion” isused to describe the fact that the method according to the presentinvention results in an increase in pH to a level of at least about 4.0,more preferably about 4.0 to about 5.0 or slightly above, in a period ofno greater than about 30 minutes, preferably in less than about 20-30minutes, even more preferably in less than about 10-20 minutes, in about15 minutes or less or alternatively, in less than about 5 minutes.

The term “secretagogue” refers to the period during which time theparental cells of the stomach secrete acid into the gastric juices tolower pH. Often the secretagogue period occurs just after a meal, butthe secretion of acid may occur at other times. The secretagogue phasecan be of short duration or longer duration.

The term gastroesophageal reflux disease or “GERD” or “acid reflux” is acondition in which the liquid content of the stomach regurgitates (backsup, or refluxes) into the esophagus. The liquid can inflame and damagethe lining of the esophagus although this occurs in a minority ofpatients. The regurgitated liquid usually contains acid and pepsin thatare produced by the stomach. The refluxed liquid also may contain bilethat has backed-up into the stomach from the duodenum. Acid is believedto be the most injurious component of the refluxed liquid. Pepsin andbile also may injure the esophagus, but their role in the production ofesophageal inflammation and damage (esophagitis) is not as clear as isthe role of acid.

GERD is a chronic condition. Once it begins, it usually is life-long. Ifthere is injury to the lining of the esophagus (esophagitis), this alsois a chronic condition. Moreover, after the esophagus has healed withtreatment and treatment is stopped, the injury will return in mostpatients within a few months. Once treatment for GERD is begun,therefore, it may be necessary to continue the treatment continually,generally for short periods of time.

Actually, the reflux of the stomach's liquid contents into the esophagusoccurs in most normal individuals. In fact, one study found that refluxoccurs as frequently in normal individuals as in patients with GERD. Inpatients with GERD, however, the refluxed liquid contains acid moreoften, and the acid remains in the esophagus longer.

Gravity, swallowing, and saliva are important protective mechanisms forthe esophagus, but they are effective only when individuals are in theupright position. At night while sleeping, gravity is not in effect,swallowing stops, and the secretion of saliva is reduced. Therefore,reflux that occurs at night is more likely to result in acid remainingin the esophagus longer and causing greater damage to the esophagus.

Certain conditions make a person susceptible to GERD. For example,reflux can be a serious problem during pregnancy. The elevated hormonelevels of pregnancy probably cause reflux by lowering the pressure inthe lower esophageal sphincter (see below). At the same time, thegrowing fetus increases the pressure in the abdomen. Both of theseeffects would be expected to increase reflux. Also, patients withdiseases that weaken the esophageal muscles (see below), such asscleroderma or mixed connective tissue diseases, are more prone todevelop reflux.

The cause of GERD is complex. There probably are multiple causes, anddifferent causes may be operative in different individuals or even inthe same individual at various times. A number of patients with GERDproduce abnormally large amounts of acid, but this is uncommon and not acontributing factor in the vast majority of patients. The factors thatcontribute to causing GERD are the lower esophageal sphincter, hiatalhernias, esophageal contractions, and emptying of the stomach.Notwithstanding the cause of GERD, the present invention may reduce thetendency of having injurious acid reflux into the esophagus, causingdamage.

When the wave of contraction in the esophagus is defective, refluxedacid is not pushed back into the stomach. In patients with GERD, severalabnormalities of contraction have been described. For example, waves ofcontraction may not begin after each swallow or the waves of contractionmay die out before they reach the stomach. Also, the pressure generatedby the contractions may be too weak to push the acid back into thestomach. Such abnormalities of contraction, which reduce the clearanceof acid from the esophagus, are found frequently in patients with GERD.In fact, they are found most frequently in those patients with the mostsevere GERD. The effects of abnormal esophageal contractions would beexpected to be worse at night when gravity is not helping to returnrefluxed acid to the stomach. Note that smoking also substantiallyreduces the clearance of acid from the esophagus. This effect continuesfor at least 6 hours after the last cigarette.

Most reflux during the day occurs after meals. This reflux probably isdue to transient LES relaxations that are caused by distention of thestomach with food. A minority of patients with GERD, about 20%, has beenfound to have stomachs that empty abnormally slowly after a meal. Theslower emptying of the stomach prolongs the distention of the stomachwith food after meals. Therefore, the slower emptying prolongs theperiod of time during which reflux is more likely to occur.

The term “non-erosive reflux disease” or “NERD” is used describe aspecific form of GERD, described above. In some cases, GERD erodes theesophageal lining, creating a condition called esophagitis. NERD is GERDthat does not cause esophagitis. Because most GERD sufferers do not haveesophagitis, NERD is the most common form of GERD. Because its namecontains the word “nonerosive,” it may appear that NERD is the leastsevere form of GERD, but this is not necessarily so. NERD is actuallymore likely to produce extra-esophageal complications, and is also lesslikely to respond to find oplication surgery. In one study, only 56% ofNERD patients (compared with 90% of patients with erosive reflux)reported that their symptoms were completely eliminated withfundoplication. NERD was also twice as likely to cause swallowingdifficulties.

Heartburn is the chief symptom of NERD. It has a number of potentialcauses, including hiatal hernia, lifestyle behaviors, and diet. Manypeople deal with heartburn by simply adjusting their behavior. In somecases, medication or surgery may be required. Traditional antacids havealso been used to treat NERD.

The term “Zollinger-Ellison syndrome” or “ZE syndrome” is usedthroughout the specification to describe a condition caused by abnormalproduction of the hormone gastrin. In ZE syndrome, small tumor(gastinoma) in the pancreas or small intestine produces the high levelsof gastrin in the blood. ZE syndrome is caused by tumors usually foundin the head of the pancreas and the upper small bowel. These tumorsproduce the hormone gastrin and are called gastrinomas. High levels ofgastrin cause overproduction of stomach acid. High stomach acid levelslead to multiple ulcers in the stomach and small bowel. Patients with ZEsyndrome may experience abdominal pain and diarrhea. The diagnosis isalso suspected in patients without symptoms who have severe ulcerationof the stomach and small bowel.

The agents of choice for treating ZE syndrome are the proton pumpinhibitors (PPI) as described hereinabove. These drugs dramaticallyreduce acid production by the stomach, and promote healing of ulcers inthe stomach and small bowel. They also provide relief of abdominal painand diarrhea.

Surgical removal of a single gastrinoma may be attempted if there is noevidence that it has spread to other organs (such as lymph nodes or theliver). Surgery on the stomach (gastrectomy) to control acid productionis rarely necessary today. Early diagnosis and surgical removal of thetumor is associated with a cure rate of only 20% to 25%. However,gastrinomas grow slowly, and patients may live for many years after thetumor is discovered. Acid-suppressing medications are very effective atcontrolling the symptoms of acid overproduction.

The term “ulcer” is used throughout the specification to describe anarea of tissue erosion, for example, especially of the lining of thegastrointestinal (GI) tract, especially of the stomach (peptic ulcer),esophagus or small intestine (duodenal ulcer). Due to the erosion, anulcer is concave. It is always depressed below the level of thesurrounding tissue. Ulcers can have diverse causes, but in the GI tract,they are believed to be primarily due to infection with the bacteria H.pyloridus (h. pylori). GI ulcers, however, may be made worse by stress,smoking and other noninfectious factors, especially including excessivestomach acid because a lower pH tends to be a better growth environmentfor H. Pyloridus.

Traditional treatments for H. pyloridus infections includeantimicrobials/antibiotics, such as amoxicillin, clarithromycin(biaxin), metronidazole (flagyl) and tetracycline (“an anti-H. pyloriagent”); H₂-blockers, such as cimetidine (tagamet), famotidine (pepcid),nizatidine (axid), ranitidine (zantac); proton pump inhibitors (PPI),such as esomeprazole (nexium), lansoprazole (prevacid), omeprazole(prilosec), pantoprazole (protonix) and rabeprazole (aciphex);cytoprotective agents, such as bismuth subsalicylate, sucralfate; andcombination agents, such as Helidac (bismuth subsalicylate,metronidazole, and tetracycline combination), Prevpac (lansoprazole,clarithromycin and amoxicillin).

The present invention may be used to treat an H. pyloridus infection ina patient by administering an effective amount of at least onepharmaceutically acceptable water-soluble zinc salt, either alone or incombination (preferably, by coadministration) with at least one other ofthe traditional treatment modalities, as described above.

The term “coadministration” or “combination therapy” is used to describea therapy in which at least two active compounds in effective amountsare used to treat one or more of the disease states or conditions asotherwise described herein at the same time. Although the termcoadministration preferably includes the administration of two activecompounds to the patient at the same time, it is not necessary that thecompounds be administered to the patient at the same time, althougheffective amounts of the individual compounds will be present in thepatient at the same time. The active compositions may include one ormore zinc salts and/or additional compounds/compositions such as protonpump inhibitors, H₂ blockers, antibiotics/antimicrobial agents,cytoprotective agents or combination agents as otherwise describedherein in effective amounts for the disease or condition for which thecompounds are typically used.

The term “proton pump inhibitor” is used throughout the specification todescribe Proton pump inhibitors as drugs that help control the painfuldiscomfort of heartburn and gastroesophageal reflux disease (GERD), andpromote the healing of stomach and duodenal ulcers. Proton pumpinhibitors are only available by prescription. They come as tablets,capsules, injections, or powders that are made into a suspension.

Proton inhibitors work by blocking the production of stomach acid. Theyinhibit a system in the stomach known as the proton pump, which isanother name for the “hydrogen-potassium adenosine triphosphate enzyme”system. Proton pump inhibitors are rather versatile. They are used toheal stomach and duodenal ulcers, including stomach ulcers caused bytaking nonsteroidal anti-inflammatory drugs. They are also used torelieve symptoms of esophagitis (inflammation of the esophagus orgullet) and severe gastroesophageal reflux (GERD), as discussed above.

Combined with certain antibiotics (such as amoxycillin andclarithromycin) or with zinc salts according to the present invention,proton pump inhibitors are effective for treating Helicobacter pyloriinfections (a bacterial infection of the stomach). The H. pyloribacterium is a chief suspect in the cause of recurring stomach ulcers.PPIs are also a first-choice treatment for the rare condition calledZollinger-Ellison syndrome, discussed above.

Proton Pump Inhibitors exhibit side effects, although they tend to bemanageable, including diarrhea, feeling or being sick, constipation,flatulence, abdominal pain, headaches and more rarely, allergicreactions, itching, dizziness, swollen ankles, muscle and joint pain,blurred vision, depression and dry mouth, among others. Long-term use ofproton pump inhibitors can result in stomach infections. Because protonpump inhibitors completely stop acid production—and stomach acid helpskill microbes such as bacteria in the stomach—using PPIs can lead togrowth of potentially harmful microbes in the stomach.

Proton pump inhibitors exhibit significant, sometimes deleterious druginteractions, including reactions with phenytoin as an epilepsy agentand warfarin to prevent blood clots, to increase their effects, withketoconazole and itraconazole to reduce their absorptivity, withdiazepam (valium) to decrease its metabolism.

Proton pump inhibitors are usually taken for 1-2 months, but in somecases may be taken longer. Symptoms may return when a person stopstaking a proton pump inhibitor. Proton pump inhibitors may causeinternal bleeding, signs of which include vomiting blood, detecting asubstance-like coffee grounds in your vomit, or pass black tarry stools,see your doctor immediately.

Common proton pump inhibitors include omeprazole (Prilosec),esomeprazole (Nexium), lansoprazole (Prevacid), pantoprazole (Protonix)and rabeprazole sodium (Aciphex).

The present invention relates to a method for providing fast action withoptional long duration effect in reducing gastric acid secretion,raising the pH of the stomach during a resting phase, decreasing theduration of stomach acid release during a secretagogue phase and fortreating conditions including gastroesophageal reflux disease (GERD),non-erosive reflux disease (NERD), Zollinger-Ellison syndrome (ZEdisease), ulcer disease, and gastric cancer where the reduction ingastric acid secretion is beneficial, as well as preventing or reducingthe likelihood of ulcer disease by reducing gastric acid section. Inaddition, the present methods are useful for treating patients who arenon-responsive to proton pump inhibitors (PPI) and as an alternative totraditional therapies or conditions which are caused by rapid andcomplete inhibition of secretagogue induced acid secretion.

The method comprises administering an effective amount of at least onepharmaceutically acceptable water-soluble zinc salt to alleviate ortreat the condition or disease state. The methods may involve theadministration of a water-soluble zinc salt alone or in combination withother agents as disclosed herein a single time, or preferably for longerduration, usually about 2-3 days to about 2-3 months, with varyingintervals in between, depending upon the prognosis and outcome of thetreatment.

Zinc salts according to the present invention may be administered aloneor in combination with other compounds, compositions or therapies,depending upon the condition or disease state to be treated, includingan effective amount of a proton pump inhibitor or other agent asotherwise described herein which may be used to treat H. pyloriinfections. These agents include proton pump inhibitors such asesomeprazole, lansoprazole, omeprazole, pantoprazole or rabeprazole, H2blockers such as cimetidine, famotidine, nizatidine or ranitidine,anti-H. pylori agents, such amoxicillin, clarithromycin (biaxin),metronidazole (flagyl) or tetracycline, cytoprotective agents such asbismuth subsalicylate or sucralfate, or a combination agent such asHelidac or Prevpac.

In a preferred aspect of the invention, at least one water-soluble zincsalt is used wherein the zinc salt or combination is characterized asbeing soluble and absorbable (through the gastrointestinal mucosa) atboth low pH (i.e., a pH of about 1-2, which occurs in an acidiccondition in the stomach) and higher pH (i.e., a pH of about 4-5 orslightly above after acid secretion in the stomach is inhibited or evenhigher—i.e., a pH of about 5.5-6.0 in the duodenum to about 6.5-7.5 inthe jejunum and ileum—the pH is slightly higher in the ileum than in thejejunum). By providing for compositions which are both water-soluble andabsorbable throughout the gastrointestinal mucosa (i.e. in the stomachand through the various sections of the small intestine), thebioavailability of the zinc salt will be maximized as will favorabletherapy of the conditions or disease states to be treated. In thisaspect, a combination of effective amounts of zinc chloride and at leastone zinc salt selected from the group consisting of zinc acetate, zincgluconate, zinc ascorbate and a zinc amino acid chelate (as mono- orbis-amino acid chelate) is preferred, although numerous other zinc acidcompounds may be combined to produce favorable results.

Preferred zinc salts include those salts in which the anionic counterionin protonated form has a pKa of at least about 4 to about 5.5 or higher.Mixtures of zinc salts wherein all of the zinc salts are soluble withina range of pH from 1-2 to about 7.5 are preferred. Zinc acetate, zincgluconate, zinc glycolate and zinc ascorbate alone or in combinationwith another zinc salt, especially zinc chloride, are particularlyuseful for use in the present invention. Zinc amino acid chelates (mono-or bis-amino acid chelates) may also be preferably used wherein acombination of zinc chloride and a zinc amino acid chelate selected fromthe group consisting of zinc chelates (mono- or bis-chelates) ofL-cysteine, L-cystine, L-N-acetylcysteine, L-histidine, D-histidine,L-taurine, L-glycinate, L-aspartate, L-methionine, and mixtures thereof.

Preferred zinc salts according to the invention include zinc chloride(where pKa of the counterion is not important because of its interactionwith chloride channels) and organic acids including zinc acetate (pka4.75), zinc gluconate, and zinc ascorbate (pka of 4.2 and 11.6). Inaddition, zinc glycolate and zinc lactate may also be used preferably,zinc glycolate being preferred. It is preferred that when a combinationof zinc salts is used that at least one zinc salt which is effective atlow pH in the stomach (for immediate inhibition of acid secretion) becombined with an agent which may exhibit a heightened effect in thestomach at a pH of 4.0-5.0 or higher, or which is preferentiallyabsorbed in the small intestine (a zinc mono- or bis-amino acidchelate).

While not being limited by way of theory, it is believed that acombination of a zinc salt which is effective at low pH in the stomach(such as zinc chloride and also zinc sulfate) and one or more of theorganic acid zinc salts which are effective at a higher pH, willmaximize delivery of zinc to the stomach mucosa to obtain a favorableeffect, at first by being dissolved in acid gastric juice in the stomachwhere an initial inhibition of acid occurs and the pH rises, andsubsequently, through absorption of zinc (from a zinc salt) at a higherpH in the stomach or in the small intestine where blood levels of zincwill increase to therapeutic levels. The absorption and effect of a zincsalt at higher pH levels in the stomach or at the higher pH of the smallintestine (5.5-7.5 or higher) is advantageous because this delayedabsorption of zinc will reduce gastric acid secretion at a later time(than an initial effect at a low pH) over an extended period of time.Compositions according to the present invention may be administered asingle time, but usually are administered preferably once or twice dailyorally for a period ranging from about 2-3 days to several months orlonger.

Compositions according to the present invention also relate to sustainedor extended release formulations which comprise a first component whichallows or facilitates fast dissolution in the gastric juices at low pHso that a rapid inhibition of acid secretion is effected (withconcombinant increase in pH to a level of about 4.0 to about 5.0 orhigher) and a second component which releases zinc salt at the higher pHlevel in the stomach or more preferably, further in the small intestineon a sustained release basis in order to maintain an effective level ofzinc in the blood stream to inhibit gastric acid secretion in thestomach for extended periods. The first fast-acting component may bereadily formulated using a zinc salt which dissolves in gastric juice atlow pH (e.g., zinc chloride or zinc sulfate at a pH about 1.0 to about2.0) using standard excipients such as lactose, confectioner's sugar inpowered form, various stearate salts, etc, which dissolves rapidly inthe stomach and a second sustained or extended release formulation whichmakes us of any number of polymeric binders, matrices (polymeric and/orerodible), granules, or enteric coatings to allow release of zinc salton an extended or sustained release basis in the small intestine. Manyof these techniques are well known in the art. Exemplary patents such asU.S. Pat. No. 4,863,741 to Becker, U.S. Pat. No. 4,938,967 to Newton, etal., U.S. Pat. No. 4,940,556 to MacFarlane, et al., and U.S. Pat. No.5,202,128 to Morella, et al., among numerous others, may be useful forproviding teachings, all well known in the art, for formulating fastrelease/sustained or extended release formulations useful in the presentinvention.

The above formulations may be useful for providing enhancedbioavailability of one or more zinc salts and optionally, other agentswhich may be useful in treating or reducing the likelihood of one ormore of gastric ulcers, GERD, NERD, Zollinger-Ellison syndrome, gastriccancer and reducing/inhibiting the secretion of acid in the stomach andraising the pH of the stomach to about 4.0 to about 5.0 or more, asotherwise disclosed in the present invention. It is noted that ininhibiting acid secretion in the stomach, blood concentrations of zincsalt of about 100 micromolar (μmol) produce inhibition of about 70%.With 300 μmol concentration of zinc salt, the inhibition approaches100%. The time of action of inhibition from the blood delivery side at100 μmol or 300 μmol is immediate (i.e., as soon as the zinc salt comesinto contact with the cell membrane, inhibition occurs. It may be shownthat inhibition occurs within about 10-15 minutes to 1 about hour in thepresence of secretagogue. The zinc salts may be administered orally(preferably no more than once or twice a day) or intravenously, alone orin combination with optional PPI drugs.

The use of zinc chloride alone, or in combination with at least oneadditional zinc salt as otherwise described herein is preferred.Additional preferred zinc salts include zinc acetate, zinc gluconate,zinc ascorbate and zinc amino acid chelates (mono- and bis-amino acidchelates). These zinc salts and combinations may be used alone or incombination with additional agents such as a proton pump inhibitor(esomeprazole, lansoprazole, omeprazole, pantoprazole or rabeprazole),an H2 blocker (cimetidine, famotidine, nizatidine or ranitidine), ananti-H. pylori agent (amoxicillin, clarithromycin, metronidazole ortetracycline), a cytoprotective agent such as bismuth subsalicylate orsucralfate, or a combination agent such as Helidac or Prevpac.

Pharmaceutical compositions comprising an effective amount of apharmaceutically acceptable zinc salt alone, or preferably incombination with at least one other zinc salt or an effective amount ofa traditional proton pump inhibitor such as such as esomeprazole,lansoprazole, omeprazole, pantoprazole or rabeprazole, an H2 blockersuch as cimetidine, famotidine, nizatidine or ranitidine, an anti-H.pylori agent, such amoxicillin, clarithromycin (biaxin), metronidazole(flagyl) or tetracycline, a cytoprotective agent such as bismuthsubsalicylate or sucralfate, or a combination agent such as Helidac orPrevpac, optionally in combination with a pharmaceutically acceptablecarrier, additive or excipient.

Pharmaceutical formulations include those suitable for oral, rectal,nasal, topical (including buccal and sub-lingual), vaginal or parenteral(including intramuscular, sub-cutaneous and intravenous) administration.Oral compositions or parenteral compositions (especially those for IVadministration) are preferred. Compositions according to the presentinvention may also be presented as a bolus, electuary or paste. Tabletsand capsules for oral administration may contain conventional excipientssuch as binding agents, fillers, lubricants, disintegrants, or wettingagents. The tablets may be coated according to methods well known in theart. Oral liquid preparations may be in the form of, for example,aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, ormay be presented as a dry product for constitution with water or othersuitable vehicle before use. Such liquid preparations may containconventional additives such as suspending agents, emulsifying agents,non-aqueous vehicles (which may include edible oils), or preservatives.When desired as discussed hereinabove, the present formulations may beadapted to provide sustained release characteristics of the activeingredient(s) in the composition using standard methods well-known inthe art. A composition which provides an effective amount of initialdose of zinc salt in the gastric juice at low pH followed by extendedrelease effects of zinc over a longer duration may be preferred.

In the pharmaceutical aspect according to the present invention, thecompound(s) according to the present invention is formulated preferablyin admixture with a pharmaceutically acceptable carrier. In general, itis preferable to administer the pharmaceutical composition orally, butcertain formulations may be preferably administered parenterally and inparticular, in intravenous or intramuscular dosage form, as well as viaother parenteral routes, such as transdermal, buccal, subcutaneous,suppository or other route, including via inhalation intranasally. Oraldosage forms are preferably administered in tablet or capsule(preferably, hard or soft gelatin) form. Intravenous and intramuscularformulations are preferably administered in sterile saline. Of course,one of ordinary skill in the art may modify the formulations within theteachings of the specification to provide numerous formulations for aparticular route of administration without rendering the compositions ofthe present invention unstable or compromising their therapeuticactivity.

In particular, the present compounds are preferably readily watersoluble and mixtures of water-soluble zincs may be used to effect animmediate release/sustained release pharmaceutical profile. This maymaximize immediate effect and longer duration effect by simply choosethe type of salt and adjusting the ratio of the zinc salt mixtureaccordingly. Of course, excipients can be chosen to affect the deliveryand bioequivalence of the zinc salts used. It is well within theroutineer's skill to modify the route of administration and dosageregimen of a particular compound in order to manage the pharmacokineticsof the present compounds for maximum beneficial effect to the patient.

Formulations containing the compounds of the invention may take the formof solid, semi-solid, lyophilized powder, or liquid dosage forms, suchas, for example, tablets, capsules, powders, sustained-releaseformulations, solutions, suspensions, emulsions, suppositories, creams,ointments, lotions, aerosols or the like, preferably in unit dosageforms suitable for simple administration of precise dosages.

The compositions typically include a conventional pharmaceutical carrieror excipient and may additionally include other medicinal agents,carriers, and the like. Preferably, the composition will be about 0.05%to about 75-80% by weight of a zinc salt compound or compounds accordingto the invention, with the remainder consisting of suitablepharmaceutical additives, carriers and/or excipients. For oraladministration, such excipients include pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharine,talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate, andthe like. If desired, the composition may also contain minor amounts ofnon-toxic auxiliary substances such as wetting agents, emulsifyingagents, or buffers.

Liquid compositions can be prepared by dissolving or dispersing thecompounds (about 0.5% to about 20%), and optional pharmaceuticaladditives, in a carrier, such as, for example, aqueous saline, aqueousdextrose, glycerol, or ethanol, to form a solution or suspension. Foruse in oral liquid preparation, the composition may be prepared as asolution, suspension, emulsion, or syrup, being supplied either inliquid form or a dried form suitable for hydration in water or normalsaline.

When the composition is employed in the form of solid preparations fororal administration, the preparations may be tablets, granules, powders,capsules or the like. In a tablet formulation, the composition istypically formulated with additives, e.g. an excipient such as asaccharide or cellulose preparation, a binder such as starch paste ormethyl cellulose, a filler, a disintegrator, and other additivestypically used in the manufacture of medical preparations.

An injectable composition for parenteral administration will typicallycontain the compound in a suitable i.v. solution, such as sterilephysiological salt solution. The composition may also be formulated as asuspension in a lipid or phospholipid, in a liposomal suspension, or inan aqueous emulsion.

The pharmaceutical compositions of this invention may also beadministered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other conventional solubilizingor dispersing agents.

Methods for preparing such dosage forms are known or will be apparent tothose skilled in the art; for example, see “Remington's PharmaceuticalSciences” (17th Ed., Mack Pub. Co, 1985). The person of ordinary skillwill take advantage of favorable pharmacokinetic parameters of thepro-drug forms of the present invention, where applicable, in deliveringthe present compounds to a patient suffering from a viral infection tomaximize the intended effect of the compound.

The pharmaceutical compositions according to the invention may alsocontain other active ingredients such as proton pump inhibitors, H₂blockers, antimicrobial agents, cytoprotective agents or combinationagents. In addition, compounds according to the present invention mayalso contain anti-cancer agents (to treat gastric cancer). Effectiveamounts or concentrations of each of the active compounds are to beincluded within the pharmaceutical compositions according to the presentinvention.

The individual components of such combinations may be administeredeither sequentially or simultaneously in separate or combinedpharmaceutical formulations.

When one or more of the compounds according to the present invention isused in combination with a second therapeutic agent active the dose ofeach compound may be either the same as or differ from that when thecompound is used alone. Appropriate doses will be readily appreciated bythose skilled in the art.

The following examples are used to describe the present invention. It isunderstood that they are merely exemplary and are understood not tolimit the breadth of the invention in any way.

EXAMPLES

The stomach produces acid to help break down food, making it easier todigest. In some cases, stomach acid can actually irritate the lining ofthe stomach and the duodenum (top end of the small intestine). Sometimesthe acid “refluxes” upwards and irritates the lining of the esophagus.Irritation of the lining of the stomach or the esophagus causes acidindigestion (heartburn) and sometimes causes ulcers or bleeding.

We show in this particular application that ZnCl₂ has a potentinhibitory effect on gastric acid secretion at the cellular level byabolishing the activity of the gastric H⁺/K⁺-ATPase in rat and humangastric glands. We also demonstrate that addition of micromoleconcentrations of ZnCl₂ can effectively prevent histamine dependent acidsecretion in whole rat stomachs and through a ZnCl₂ enriched diet.

Material and Methods

Animals. Sprague-Dawley rats 150-250 g (Charles River Laboratory)) werehoused in climate- and humidity-controlled, light-cycled rooms, fedstandard chow with free access to water, and handled according to thehumane practices of animal care established by the Yale Animal Care.Prior to experiments, animals were fasted for 18-24 hours with freeaccess to water.

Isolation of rat and human gastric Glands. Following removal of thestomach, the stomach was opened longitudinally and the corpus and antrumisolated and sliced into 0.5 cm square sections, and washed with coldRinger solution to remove residual food particles. The tissues weretransferred to the stage of a dissecting microscope. Individual glandswere isolated using a hand-dissection technique as describedpreviously³⁶. Following isolation, individual isolated glands wereallowed to adhere to cover slips that had been pre-coated with Cell-Tak(Collaborative Research, Bedford, Mass.) and were transferred to thestage of an inverted microscope.

The human tissue was transferred from the OR in a HEPES-buffered Ringersolution. The tissue was stored on ice and immediately the isolatedglands were dissected as described above.

Digital imaging for intracellular pH. Isolated gastric glands wereincubated in a HEPES-buffered Ringer's solution containing either 10μmol of the pH-sensitive dye BCECF-AM(2′,7′)-bis-(2-carboxyethyl)-5-(and-6)-carboxy-fluorescin, aceto-methylester (Molecular Probes, Eugene, Oreg.) for 10 minutes as describedpreviously³⁷⁻³⁹. Following dye-loading the chamber was flushed with aHEPES solution to remove all non-de-esterfied dye. The perfusion chamberwas mounted on the stage of an inverted microscope (Olympus IX50), whichwas used in the epifluorescence mode with a 40× objective. BCECF wassuccessively excited at 440 nm and 490 nm from a monochromator lightsource, and the resultant fluorescent signal was monitored at 535 nmusing an intensified charge-coupled device camera. Individual regions ofinterest were outlined and simultaneously monitored every 15 sec. duringthe course of the experiment. A minimum of 8 cells or regions wasselected per gland.

Proton extrusion by individual parietal cells was monitored by observingrecovery of pH_(i) after acid loading the cells with Na⁺ free HEPESsolution containing 20 mM NH₄Cl. Parietal cells were subsequentlysuperfused with Na⁺ free HEPES, which abolished all Na⁺/H⁺ Exchanger(NHE) activity, trapping H⁺ within the cytosol and initiating animmediate drop in pHi. Under these conditions, the only potential H⁺extrusion pathway is via the H⁺/K⁺-ATPase activation.

The intensity ratio data (490/440) were converted to pH values using thehigh K+/nigericin calibration technique⁴⁰. Intracellular pH recoveryrates were calculated from the same initial starting pH to eliminate thepotential variation in the individual intracellular buffering power ofthe cells under the different experimental conditions. All dataincluding the individual images for all wavelengths were recorded to thehard disk which allowed us to return to the individual images after theexperiment for further analysis. The recovery rates are expressed as theΔpH/min, and were calculated over the pH range of 6.5-6.8. All chemicalswere obtained from Sigma and Molecular Probes. All data were summarizedas means±SE and were analysed by grouping measurements at baselinevalues.

Whole stomach pH measurements. Before the experiments animals werefasted for 24 h to reduce basal acid secretion to a consistent minimum.Animals were killed with an overdose of isoflurane and an abdominalincision was made. The stomach was ligated at the duodenal andesophageal junction and excised. Then 1 ml of non buffered, isotonicsaline (140 mM) was infused into the lumen of the stomach. This volumedid not distend the stomach, thus avoiding potential stimulation of acidsecretion by stretch. The stomachs were then placed in either oxygenatedHEPES-buffered Ringer solution or in the same solution containing 100 μMhistamine alone or additionally 300 μM ZnCl₂ (pH 7.4) and maintained at37° C. After 1 hour the stomach contents were aspirated and the pH wasrecorded.

Oral Zinc supplementation in rats. These studies were designed tomodulate acid secretion by increasing dietary zinc. In these studies weused an oral ZnCl₂ solution (zinc chloride in tap water). The animalshad free access to food and the zinc containing water for the durationof the study. 150 mg/kg/d or 0.5 mg/kg/d ZnCl₂ was added to the drinkingwater for 5 days. Animals had free access to water prior to theexperiment and were fed with standard chow until 24 hours before theexperiment, at which point they had free access to ZnCl₂ containingwater only. After the 5 days exposure period and the 24 hour fast, theanimals were sacrificed and a total gastrectomy was performed on theanimals. Individual gastric glands were isolated with the handdissection technique described above.

Results

Histamine induced acid secretion in human and rat is inhibited by ZnCl₂.In the first series pH_(i) measurements of single parietal cells withinfreshly isolated gastric glands were used to measure H⁺/K⁺-ATPaseactivity. The activity of the proton pump was calculated from the rateof alkalinization of pH_(i) (ΔpH_(i)/min) after acidification using theNH4Cl prepulse technique in the absence of sodium and bicarbonate. H⁺extrusion under these conditions depends on the activity of theH⁺/K⁺-ATPase, as previously shown⁴¹. In the absence of any stimulation,only a low rate of pHi recovery was observed (0.011±0.002 pH_(i)units/min, n=32 cells from 3 glands from 3 animals; FIG. 1E). Afterexposure of the rat gastric glands to histamine (100 μM), thealkalinization rate increased to 0.051±0.004 pH units/min (n=60 cellsfrom 15 glands from 8 animals; FIG. 1A). Adding 300 μM ZnCl₂ to thesuperfusion bath in the presence of histamine (100 μM) prevented thestimulatory effect of histamine on the Na⁺-independent pH_(i) recoveryrate (0.0012±0.004 pH units/min) and reduced it to the same level asseen in the control glands not exposed to histamine (n=60 cells from 6glands from 4 animals); FIG. 1B). Human gastric glands showed also arobust proton efflux under histamine stimulation. This effect wasabolished by ZnCl₂ (FIG. 1C, D); (n=26 cells, 3 glands). Thus thefreshly isolated rat and human gastric glands showed H⁺/K⁺-ATPaseactivity that could be stimulated by histamine and inhibited by ZnCl₂.

ZnCl₂ inhibits rat acid secretion in a dose dependent manner. ZnCl₂inhibited H⁺ extrusion in a dose dependent manner (FIG. 2). In thisprotocol acid secretion was stimulated by histamine and expressed asΔpH_(i)/min. Therefore rat gastric glands were incubated with 100 μMhistamine (15 min) and histamine was present throughout the entireexperiment. To investigate the inhibitory potency of ZnCl₂ we useddifferent concentrations (25 μM-300 μM). ZnCl₂ was present during theentire experiment, including the histamine incubation period of 15 min.300 μM ZnCl₂ showed a 98% inhibition of proton extrusion compared to theHistamine induced rate and the control.

Fast onset and reversible inhibition of gastric acid secretion by ZnCl₂.There are irreversible (i.g. omeprazole) and reversible (P-CAB's) acidblockers available⁴². We investigate the reversibility of the inhibitoryeffect of ZnCl₂ in our in vitro setting. Thus we stimulated acidsecretion with histamine (100 μM) during the entire experiment. When theintracellular alkalinization (acid secretion) was observed, ZnCl₂ (300μM) was added to the superfusion bath. The acid secretion was abolishedafter a few seconds (FIG. 3A). After the removal of ZnCl₂ in the sameexperiment acid secretion returns to normal levels. We were also able todemonstrate reversibility following incubation and superfusion ofparietal cells over 20 min with ZnCl₂ (300 μM) and histamine (100 μM).After removal of ZnCl₂ from the superfusion bath the intracellularalkalinization (proton extrusion) proceeded at normal uninhibited rate(FIG. 3B).

Oral Zinc supplementation reduces basal rat gastric acid secretion.These studies were designed to modulate acid secretion by increasingdietary zinc. 150 mg/kg/d or 0.5 mg/kg/d ZnCl₂ were added to thedrinking water for 5 days. The H⁺ extrusion rate was measured with BCECFas described before. Histamine stimulated parietal cells showed a robustrecovery rate (proton extrusion) of 0.051±0.004 (n=120 cells from 15glands from 8 animals). FIG. 4 shows that the ZnCl₂ (150 mg/kg/d or 0.5mg/kg/d) in the drinking water decreased the histamine induced acidsecretion significantly in comparison to the control group withhistamine alone. 150 mg/kg/d: 0.022±0.0045; (n=60 cells from 10 glandsfrom 3 animals), 0.05 mg/kg/d: 0.034±0.0036; (n=60 cells from 6 glandsfrom 4 animals).

ZnCl₂ decreased gastric acid production ex vivo. To determine whetherZnCl₂ could inhibit gastric acid secretion in the whole organ, weexamined luminal pH in freshly isolated rat stomachs after incubation inHEPES or in the same solution the presence of 100 μM histamine or both,100 μM histamine and 300 μM ZnCl₂. As illustrated in FIG. 5, in thepresence of histamine mean luminal pH was lower than in control stomachpreparations incubated in HEPES alone (3.15±0.27 vs. 4.59±0.48, n=9 foreach, P<0.005). In the presence of histamine and ZnCl₂ the luminal pHwas nearly as high as in the control group without stimulation althoughthis findings were not significant (4.54±0.065 vs. 4.59±0.48, n=8 eachgroup, P>0.005).

Different Zinc Salts shows different efficacy in raising intraluminalpH. Measurements of whole stomach intraluminal pH using a number of zincsalts according to the present invention were made to assess effect ofsalt and concentration on intraluminal pH. Isolated whole stomachpreparations from rats were cannulated at the esophageal and duodenaljunction and perfused in vitro with 37° C. pH 7.4 Ringers solution. Theblood perfusate was then exposed to 100 μM Histamine to induce acidsecretion. The lumen of the stomach was infused with 0.5 cc ofnon-buffered isotonic saline. In some studies one of the following zincsalts was added to the lumen perfusate at a final concentration of 300μM (zinc chloride, zinc sulfate, zinc acetate, zinc citrate). The dataare the sum of 5 separate stomachs from 5 separate animals for each ofthe columns. Data are the mean of all studies with the standard error ofthe mean displayed. Those results appear in attached FIG. 6.

Discussion

In this study, we examined the dose dependent inhibition of gastric acidsecretion by ZnCl₂ in human and rat gastric glands. Furthermore we triedto evaluate the onset of effect of ZnCl₂ and used whole stomachpreparation as well as oral Zinc supplementation to investigate theeffect on gastric acid secretion.

Acid secretion was induced by the classically known secretagoguehistamine, which led to a robust proton extrusion via the H⁺,K⁺-ATPasein comparison to basal acid secretion in the resting, unstimulated gland(FIG. 1e ). In subsequent studies we examined the inhibitory effects ofZnCl₂ on secretagogue sensitive gastric acid secretion. We confirmed theinhibitory potency of ZnCl₂ (300 μM) on histamine induced acidsecretion. ZnCl₂ inhibits acid secretion in the single gastric gland ina dose dependent manner. ZnCl₂ abolished proton extrusion to a levelcomparable to that of the control experiments in both, human and ratgastric glands (FIG. 1). This dose would be equivalent to 40 mgsupplementation per day in humans. The daily recommended amount of Zincintake is 11 mg. In the literature the amount considered to be toxic is10 times higher. Therefore 40 mg of ZnCl₂ as an oral acid blocker wouldbe significantly lower than reported toxic doses. In addition a similaramount of ZnCl₂ also prevented acid secretion in ex vivo whole stomachpreparations (FIG. 5). In these experiments ZnCl₂ was applied to theluminal side of the stomach and it can thus be concluded that the metalion is working directly on the H⁺/K⁺-ATPase of the parietal cell orenters the cell to modulate the signalling pathway of acid secretion. Itremains unclear how ZnCl₂ enters the cell. Previous studies describedZinc entry into the cell through voltage dependent Ca²⁺-channels and/orthe HCO₃/Cl⁻ exchanger on the basolateral membrane. Orally applied ZnCl₂confirmed our previous results. Proton extrusion by ZnCl₂ treated ratswas significant lower than acid secretion by our control group (FIG. 4).The control refers to histamine stimulated. In the figure, zinc treatedglands are still higher than the control (non-histamine treated) alone.

As mentioned in the introduction proton pump inhibitors have a delayedonset of acute action and the full inhibitory effect is slow requiringseveral dose cycles. For example omeprazole reaches only 30% inhibitionof acid secretion on the first day of treatment⁴³. Our studycharacterizes the rapid onset of action ZnCl₂ as well as itsreversibility. Faster onset of effect and increased duration of actionwould offer improvement for patients with GERD and other acid relateddisorders. In fact as shown in FIG. 5a we were able to inhibit histamineinduced acid secretion during maximal proton extrusion by addition of300 μM ZnCl₂. On the other hand histamine induced acid secretioncontinued after removal of ZnCl₂ from the superfusion bath demonstratingthe reversible nature of ZnCl₂ (FIG. 3b ).

In summary our findings indicate that ZnCl₂ offer a more rapid andprolonged inhibition of gastric acid secretion. It is a reversible andfast acting inhibitor of acid secretion in single rat and human gastricglands and also in whole stomach preparations.

Such treatment may provide significant benefit to patients with GERD.Future studies investigating the exact mechanism by which ZnCl₂ inhibitsacid secretion are necessary and will help define its future place inthe treatment of acid related diseases in the clinical setting.

Fundic Region

In the following examples, it is shown that the fundic region of thestomach and the fundic glands contain functional acid secretoryproteins. Furthermore, it is shown that the fundic glands have a sodiumand potassium independent protein the proton ATPase commonly referred toas the Vacuolar H⁺-ATPase. The evidence consists of immunofluorescencedata using a antibody directed against the a Subunit of the H⁺-ATPaseand functional data (FIGS. 7-10) in which the extrusion rate of protonsfrom these cells in the absence of Na and K is measured. Further thereis evidence that this process is amplified in the presence of histaminea compound that was thought to only influence the gastric H,KATPasefound in the parietal cells in the body of the stomach. This activity isdemonstrated in both the rat model and in humans in gastric resectionstaken from patients undergoing gastric reduction surgery.

Materials and Methods

Animals and Chemicals

Male Sprague Dawley rats weighing 200-300 g were housed in climate andhumidity controlled, light cycled rooms and fed standard chow with freeaccess to water. Prior to experiments, animals were fasted whileallowing free access to water for 18-24 hours to reduce basal acidsecretion. Following isoflurane anesthesia the animals were sacrificed,an abdominal incision was made exposing the stomach. After isolating theesophagus and duodenal junctures a total gastrectomy was performed with1-2 cm of esophagus remaining attached to the gastrectomy. We includedthe esophageal juncture to have a common landmark for all fundicisolations. While holding the esophagus with forceps approximately 3 mmof fundus was removed with 5 mm of the intact esophagus.

Fundic Gland Isolation

The removed fundic tissue was placed in ice cold HEPES-bufferedRinger-solution (pH adjusted to 7.4 at 4° C.) and transferred to thestage of a dissection microscope. The fundic glands were visualizedunder the microscope at 50× magnification. Glands adjacent to theesophageal junction were hand dissected. Following isolation, individualglands were adhered to cover slips that had been pretreated with thebiological adhesive Cell-Tak (Cell-Tak™ cell adhesive, BD Biosciences;Bedford, Mass.)

Immunohistochemistry/Immunofluorescence

Male Wistar rats (200-250 g) were anesthetized with pentobarbital i.p.and perfused through the left ventricle with PBS followed byparaformaldehyde-lysine-periodate (PLP) fixative as previouslydescribed^(19a). Stomachs were removed, cleaned from food residues, andfixed overnight at 4° C. by immersion in PLP. Stomachs were washed threetimes with PBS and sections were cut at a thickness of 5 μm aftercryoprotection with 2.3 M in PBS for at least 12 h. Immunostaining wascarried out as described previously^(20a). Sections were incubated with1% SDS for 5 min., washed 3 times with PBS and incubated with PBScontaining 1% bovine serum albumin for 15 min prior to the primaryantibody. The primary antibodies (mouse monoclonal anti human β gastricH⁺, K⁺-ATPase (Affinity Bioreagents, CA, USA) diluted 1:50 in PBS andapplied overnight at 4° C. Sections were then washed twice for 5 minwith high NaCl PBS (PBS+2.7% NaCl), once with PBS, and incubated withthe secondary antibody (donkey anti-rabbit Alexa 546, Molecular Probes,Oregon) at a dilution of 1:1000 for 1 h at room temperature. Sectionswere washed twice with high NaCl PBS and once with PBS before mountingwith VectaMount (Vector Laboratories, Burlingame, Calif.). The specimenswere viewed with a Nikon E-800 microscope.

ImmunoGold Labeling

Rats were anaesthetized using 5 ml of a 10% sodium pentobarbital giveni.p. Fixation was done through a left ventricle cardiac perfusion usingPBS and then PLP. The stomach was removed and fixed in PLP for 4 hoursand then transferred to holding buffer overnight. Frozen and Eponsections of the gastro esophageal junction were made and slices weretaken for gold labeling and electron microscopy.

Hematoxylin and Eosin Staining

Rats were anaesthetized using 5 ml of a 10% sodium pentobarbital giveni.p. Fixation was done through a left ventricle cardiac perfusion usingPBS to flush the animal and then Karnovsky Fixative for 2 hours and thenin holding buffer overnight. A section of the gastro esophageal junctionwere made and slices taken for Electron Microscopy morphology of the H⁺,K⁺-ATPase protein and Hematoxylin/Eosin staining of the glands at thegastro esophageal junction.

Measurements of Intracellular pH (pH_(i)) Measurements of IsolatedFundic Glands

Using the same protocol that we developed for isolated corpus glandperfusion^(20a, 21a) individual fundic glands were loaded with a 10 μMconcentration of the pH sensitive dye (BCECF,(2′7-bis(2-carbocyethal)-5-(and 6)-carboxyflurorescein-acetomethylester;Molecular Probes, OR USA)) for 15 minutes. Following the loading period,the perfusion chamber was mounted on the stage of an inverted microscope(Olympus IX50) attached to a digital imaging system (Universal ImagingCorp; Dowingtown, Pa.), and perfused with HEPES buffered Ringer-solutionfor 5 min at 37 C° to remove any unesterified dye. Measurements wereperformed in the epifluorescence mode with 60×/0.80 and 40×/0.90objectives. BCECF was successively excited at 440±10 nm and 490±10 nm,the resultant intracellular fluorescent signal was monitored at 535 nmusing an intensified charge-coupled device camera. Data points wereacquired every 15 s. The resulting 490/440 intensity ratio data wereconverted to intracellular pH (pH_(i)) values using the highK⁺/Nigericin calibration technique^(22as, 22as). Acid extrusion wasmonitored in the absence of bicarbonate. The rate of intracellularalkalinization was measured after using the NH₄Cl-prepulsetechnique^(22a, 23a), which resulted in a reproducible and sustainedintracellular acidification. Intracellular pH recovery rates (H⁺,K⁺-ATPase activity) were measured in Na⁺ free HEPES solutionscontaining: 1) 100 μM histamine 2) 100 μM pentagastrin. 3) 100 μMacetylcholine 4) 100 μM histamine+omeprazole at 100 μM and 200 μMconcentrations.

Intracellular pH recovery rates were calculated from the same initialstarting pH to eliminate the potential variation in the individualintracellular buffering power of the cells under the differentexperimental conditions. All data including the individual images forall wavelengths were recorded to the hard disk which allowed us toreturn to the individual images after the experiment for furtheranalysis. The recovery rates are expressed as the ΔpH_(i)/min, and werecalculated over the pH range of 6.5-6.9.

Activation of acid secretion via histamine, acetylcholine orpentagastrin was induced by preincubation of the glands for 15 minbefore the experiment combined with BCECF (100 μM) loading. All data aresummarized as mean±S.E. Significance was determined using the one-wayANOVA test with p<0.05 considered to be statistically significant. Allchemicals used were obtained from Sigma and Molecular Probes.

Results

Immunohistochemical Localization of the H⁺, K⁺-ATPase

Immunohistochemistry using specific antibodies directed against highlyconserved epitopes within either the α or β subunits of the gastric H⁺,K⁺-ATPase identified specific staining for both subunits in the fundicglands (FIG. 7A).

Electron Microscopy

After obtaining Epon sections of fasted rat gastro esophageal junctionElectron Microscopy was done on the gastric glands that came right afterthis junction and those we named F1 and used in all of our experiments.FIG. 7B, C shows the gold tag localization to the H⁺, K⁺-ATPase in theparietal cell from the Fundic gland. We noticed a higher density ofstaining on the apical pole of the cell in the secretory or vacuolarcanaliculi. This may correlate with the fundic regions high basal protonextrusion rates in comparison to the corpus due to the fact that theprotein is always at the membrane in the fundic gland whereas in thecorpus the receptor is inside the secretory canaliculus untilstimulation.

H2 Receptor Staining

H2 receptor staining was done on both the fundus and corpus to examinethe presence and density of the receptor in both the areas of thestomach. We found clear basolateral staining in the corpus glands andcould not detect staining in the fundic glands. These results correlatewith the lack of effect by histamine in stimulating fundic acidsecretion. It was clearly seen that the H2 receptor is absent in theglands of the fundus and present in the corpus (data not shown)

Secretagogue Induced Acid Secretion

Intracellular pH was measured using the pH sensitive dye BCECF andmonitored continuously using a real time fluorescence imaging system toidentify changes in intracellular pH. Rates of proton efflux werecalculated as ΔpH_(i)/min using a technique that was developed in ourlaboratory for corpus glands^(21a-25a). We measured the change in rateof efflux in the presence and absence of secretagogues.

Histamine Effect on Fundic and Corpus H⁺, K⁺-ATPase

We incubated individual glands with 100 μM histamine for 20 minutes.Histamine was present during the whole superfusion protocol. In thecorpus gland we measured a histamine stimulated proton extrusion rate of0.056±0.008 ΔpH_(i)/min whereas the basal acid secretion without anysecretagogues was 0.011±0.002 ΔpH_(i)/min (FIG. 8C, D). In comparison tothe corpus the fundus showed even under basal conditions a high protonextrusion rate (0.039±0.009 ΔpH_(i)/min). This is similar to thehistamine induced acid secretion (0.04±0.0079 ΔpH_(i)/min, FIG. 8A, B)This data shows that there is no effect of histamine on F1 zone glandsin comparison to controls.

Acetylcholine and Fundic Acid Secretion

In the next series we investigated the functional properties of fundicglands according to the neuronal stimulation via ACH. In contrast tohistamine there was a noticeable change in proton extrusion rates afterstimulation. Although the controls were still actively pumping outprotons the glands that were stimulated with 100 μM of acetylcholine for20 minutes during the dye loading and throughout the perfusion. Wedetermined that acetylcholine caused an increase in the rate ofalkalinization (0.075±0.0015 ΔpH_(i)/min vs. controls 0.039±0.009ΔpH_(i)/min) showing a direct effect of acetylcholine on fundic acidextrusion (FIG. 9).

Pentagastrin Effect on the F1 Zone

To determine if gastrin could also activate the fundic H⁺, K⁺-ATPase weconducted studies using pentagastrin, a synthetic peptide containing theentire five terminal amino acids of gastrin, which is know to causerobust acid secretion in corpus glands. At a dose of 100 μM pentagastrinwe observed alkalinization rates that were 0.062±0.007 ΔpH_(i)/min whichwas similar to acetylcholine in terms of enhancing the rate of protonextrusion from fundic cells (FIG. 9).

Inhibitors of Gastric Acid Secretion

In the next series of studies we tried to determine if the fundic glandshad similar H⁺, K⁺-ATPase inhibitor profiles as observed in the corpus.We chose the well characterized inhibitor of the gastric H⁺, K⁺-ATPaseomeprazole and the P-CAB (potassium competitive acid blocker)AZD0865^(26, 27).

Omeprazole Effect on the F1 Zone and Corpus

As shown in FIG. 10A, omeprazole did not inhibit acid secretion usingthe same concentration that completely inhibited secretagogue inducedacid secretion in the corpus (FIG. 10B). At even a higher dose than whatnormally inhibits acid secretion in the corpus the fundus continued toextrude protons. The fundic glands were preincubated with 200 μMomeprazole and 100 μM histamine and then perfused with omeprazole andhistamine throughout the entire experiment. Alkalinization rates were0.045±0.002 ΔpH_(i)/min compared to only histamine stimulated controlsat a rate of 0.042±0.007 ΔpH_(i)/min. In contrast acid secretion in thecorpus glands was abolished by 200 μmol omeprazole (0.014±0.002ΔpH_(i)/min.), (FIG. 10B).

AZD0865 Effect on the F1 Zone in Comparison to the Corpus:

Also shown in FIG. 10(C, D), P-CAB AZD0865 effectively inhibits acidsecretion in the corpus at a 10 μM concentration; however at that sameconcentration the F1 zone still has potassium dependant recovery. In thefundus the intracellular pH increased at a rate of 0.031±0.006ΔpH_(i)/min. In the Corpus at the same concentration of 10 μM therecovery rate was 0.021±0.008 ΔpH_(i)/min.

Table 1

Composition of solutions used for intracellular pH measurements insingle rat gastric glands. All concentrations are given in Mm. NMDG isN-Methyl-D-Glucosamine, all solutions were titrated to pH 7.4 at 37° C.using either NaOH or KOH. NMDG was titrated with HCL.

TABLE 1 Solution 3: Solution 2: Na⁺-free Solution 4: Solution 1:Na⁺-free HEPES + High K⁺ Standard HEPES HEPES NH4Cl calibration NaCl 125— — — NMDG — 125 125 125 NH₄Cl — — 20 — KCl 3 3 3 105 MgSO₄ 1.2 1.2 1.21.2 CaCl₂ 1 1 1 1 Glucose 5 5 5 — HEPES 32.2 32.2 32.2 32.2 pH 7.4 7.47.4 7.0Discussion

In this study we have provided evidence that the fundic region of thestomach contains glands that are capable of secreting acid via thegastric H⁺, K⁺-ATPase. In our study we have for the first timecharacterized the acid secretory properties of the fundus. We providemorphological, immunohistochemical and functional evidence for H⁺,K⁺-ATPase protein activity in the fundus In our morphological studies wefirst had to delineate where the fundus began and ended. As it isunderstood that this region begins at the gastro esophageal junction wedecided to take glands from this junction point until the initiation ofthe greater curve of the fundus. We took tissue sections from what wecalled the F1 zone starting from the gastro esophageal junction andcontinued 2 mm distally. We found these glands to be quite different inshape and also in parietal cell like density. To confirm that parietalcells in F1 contain H⁺, K⁺-ATPase we stained for the α and β subunit(FIG. 7 shows the staining for the α subunit).

During our investigation of secretagogue induced fundic acid secretionwe were able to demonstrate that histamine is not the most potentstimulator of acid secretion in the fundus as it is in the corpus. Infact, very little difference was seen in glands that were not stimulatedcompared to those stimulated with histamine (FIGS. 8 and 9). This resultwas confirmed by a lack of staining for the H2 receptor in the fundus(data not shown). Acetylcholine was the most robust of the threesecretagogues in the fundus, which may relate to the close proximity ofthe vagal nerve to the fundic region. As this section of the stomachstretches when food is present, there is vagal stimulation^(28a-30a)with associated acetylcholine secretion. This finding is especiallyimportant when considering clinical problems in obese patients who havegastro esophageal reflux disease (GERD). This can be correlated to thebenefits patients with sever ulcer disease gain when undergoing avagotomy after not gaining relief from medical management^(31a-33a).

Another interesting finding is the lack of inhibition by omeprazole onfundic acid secretion although they are immunoreactive with antibodiesfor the gastric H⁺, K⁺-ATPase. We were unable to inhibit basal orsecretagogue (histamine) induced fundic acid secretion with the protonpump inhibitor omeprazole (FIG. 10A) at doses that were double thatwhich effectively eliminated all acid secretion in the corpus (FIG.10B). These findings are in direct contrast to our data (FIG. 8C,D)^(20a, 21a, 24a, 25a) and others findings in glands from thecorpus^(34a-38a) This finding has an interesting clinical correlate inthat there is an increasing number of patients suffering from GERD thatare not effectively treated with PPI's.^(39a) One possible explanationfor the lack of omeprazole sensitivity could be that as omeprazole needsto be acid activated, a lack of a canaliculi like space would preventthe concentration of acid and potentially prevent the acid activation ofthe drug. As shown in FIG. 7B, C using immunogold tag labeling we seethat there is indeed a secretory space and that the pumps appear to linethe apical surface of this space. With previous theories of acidsecretion came the many dictums for surgical and medical modalities oftreatment which focused on the corpus. Of interest is the recentincidence of GERD like symptoms in gastric bypass patients who followingthe procedure are left only with a small part of the fundus, and littleto no functional corpus. In those symptomatic patients there has beenlittle to no success using classical PPI's^(40a) which now can bepossibly be explained by our recent findings. Our findings demonstratethat the fundic region of the stomach is much more than a holding areaand in fact can secrete acid in response to secretagogue stimulation,furthermore the H⁺, K⁺-ATPase found in this segment appears insensitiveto omeprazole. These results can lead to important new targets forpatients that are PPI resistant or have recurrent reflux symptoms in thepresence of PPI therapy.

Results:

Fundic glands showed a distinct morphology compared to corpus glands(elongated and lacking typical bulging parietal cells).Immunofluorescence (α and β subunit of the H⁺, K⁺-ATPase) and immunogoldlabeling (β subunit) were both positive in the fundic region. Fundicgland proton extrusion rates were stimulated by gastrin andacetylcholine but were not influenced by histamine. Finally acidsecretion of stimulated fundic glands could not be inhibited by the H⁺,K⁺-ATPase inhibitor omeprazole.

CONCLUSION

The fundic region of the stomach secreted acid via the H⁺, K⁺ ATPase,and was not sensitive to proton pump inhibitors. Our findingsdemonstrate that the fundic region of the stomach is much more than aholding area and in fact can secrete acid in response to secretagoguestimulation, except histamine.

The digestion of food by the stomach requires a complex combination ofhormonal and neuronal events. Generally it has been thought that thecorpus or body of the stomach secretes acid via the parietal cells andthe antrum secretes bicarbonate to neutralize the digestate by raisingthe pH of the stomach contents^(1a-9a). During this process theperistaltic movements of the stomach result in contractions that pushthe food upward into the fundic section of the stomach where it transitsbefore exiting into the small intestines^(10a). In this model ofdigestion the fundus acts only as a holding zone and is not involved inacid secretion^(4a, 11a). Classical gastric acid secretion in the corpusoccurs when the H⁺, K⁺-ATPase gets stimulated by secretagogues andbegins to secrete protons into the secretory canaliculus after beingtrafficked to the apical membrane from their cytoplasmictubulovesicles^(12a). The parietal cell has at least three activatingreceptors on its basolateral membrane, i.e. histamine H2, acetylcholineM3 and Gastrin CCK-B. It is well accepted that the H2 receptor couplesto Gs to activated adenylate cyclase producing cAMP and subsequentactivation of cAMP dependant protein kinase. The acetylcholine andgastrin receptor couple through a non Gs system probably Gq to activatephospholipase C producing IP3 and diacylglycerol. Acetylcholine releasesintracellular Ca²⁺ and gastrin activating protein kinase C^(13a). Afterthis cascade of intracellular events the parietal cell extrudes protonsvia the H⁺, K⁺-ATPase pump which exchanges intracellular H⁺ ions forextracellular K⁺ ions in an electroneutral ratio^(14a).

Recent observations in patients who have undergone gastric bypasssurgery present an interesting paradigm, namely that only left with asmall fundic region postoperatively, they still have acid secretionwhich in some patients leads to reflux symptoms, ulcers and entericcontent leaks^(15a). Of note is that many of these patients have littlesuccess in abating the symptoms while on proton pump inhibitor (PPI)therapy^(16a-18a). From these initial clinical observations we raisedthe question: does the fundus play a role in the production of acid, andhow similar are its properties to corpus secretory proteins. We alsowere interested in determining fundic sensitivity to classicalsecretagogues and therefore conducted studies using histamine,pentagastrin, and acetylcholine.

In the present experiment we have investigated the acid secretoryproperties of the rat fundus under resting and secretagogue stimulatedstates, furthermore we elucidate fundic response to an inhibitory drugof acid secretion. Our data demonstrate that the fundic region is anactive secretory zone in the stomach and contains a gastric H⁺,K⁺-ATPase that can be stimulated by secretagogues but appears to beinsensitive to omeprazole. Zinc therapy according to the presentinvention is a means to regulate acid release in the fundus region.

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The invention claimed is:
 1. A pharmaceutical composition adapted for oral administration to gastric juices in the stomach of a human patient in need of a rapid increase in gastric pH consisting essentially of an effective amount of a proton pump inhibitor and at least one pharmaceutically acceptable zinc salt selected from the group consisting of zinc acetate, zinc chloride, zinc gluconate, zinc lactate, zinc picolinate, zinc tartrate and mixtures thereof formulated for direct delivery of said zinc salt and said proton pump inhibitor to and dissolution in the gastric juices from said composition to rapidly increase the pH of the gastric juices in the stomach of said patient in need thereof to at least 3.0 within a period no greater than one hour after oral administration, wherein said composition further includes a pharmaceutically acceptable carrier, additive or excipient, and said composition excludes a buffering agent and additional compounds that increases the gastric pH.
 2. The composition according to claim 1 wherein said amount of zinc salt in said composition is effective to raise the pH of the gastric juices in the stomach of said patient to at least 3.5 within a period no greater than 30 minutes after administration.
 3. The composition according to claim 1 wherein said amount of zinc salt is effective to raise the pH of the gastric juices in the stomach of said patient to at least 4.0.
 4. The composition according to claim 1 wherein said zinc salt is zinc acetate.
 5. The composition according to claim 1 wherein said zinc salt is zinc chloride.
 6. The composition according to claim 1 wherein said zinc salt is zinc gluconate.
 7. The composition according to claim 1 wherein said zinc salt is zinc lactate.
 8. The composition according to claim 1 wherein said zinc salt is zinc picolinate.
 9. The composition according to claim 1 wherein said zinc salt is zinc tartrate.
 10. The composition according to claim 1 wherein said zinc salt is a mixture of said zinc salts.
 11. The composition according to claim 10 wherein said mixture of zinc salts includes zinc acetate.
 12. The composition according to claim 10 wherein said mixture of zinc salts includes zinc chloride.
 13. The composition according to claim 10 wherein said mixture of zinc salts includes zinc gluconate.
 14. The composition according to claim 10 wherein said mixture of zinc salts includes zinc lactate.
 15. The composition according to claim 10 wherein said mixture of zinc salts includes zinc picolinate.
 16. The composition according to claims 1 wherein said proton pump inhibitor is esomeprazole, lansoprazole, omeprazole, pantoprazole, rabeprazole or mixtures thereof.
 17. The composition according to claims 2 wherein said proton pump inhibitor is esomeprazole, lansoprazole, omeprazole, pantoprazole, rabeprazole or a mixture thereof.
 18. The composition according to claims 3 wherein said proton pump inhibitor is esomeprazole, lansoprazole, omeprazole, pantoprazole, rabeprazole or a mixture thereof.
 19. The composition according to claim 1 wherein said proton pump inhibitor is esomeprazole.
 20. The composition according to claim 2 wherein said proton pump inhibitor is esomeprazole.
 21. The composition according to claim 3 wherein said proton pump inhibitor is esomeprazole.
 22. The composition according to claim 1 wherein said proton pump inhibitor is omeprazole.
 23. The composition according to claim 2 wherein said proton pump inhibitor is omeprazole.
 24. The composition according to claim 3 wherein said proton pump inhibitor is omeprazole.
 25. The composition according to claim 3 wherein said proton pump inhibitor is lansoprazole.
 26. The composition according to claim 3 wherein said proton pump inhibitor is pantoprazole.
 27. The composition according to claim 3 wherein said proton pump inhibitor is rabeprazole.
 28. The composition according to claim 3 wherein said proton pump inhibitor is a mixture of at least two compounds selected from the group consisting of esomeprazole, lansoprazole, omeprazole, pantoprazole and rabeprazole.
 29. The composition according to claim 10 wherein said mixture of zinc salts includes zinc tartrate.
 30. The composition according to claim 2 wherein said zinc salt is at least one zinc salt selected from the group consisting of zinc acetate, zinc chloride and zinc gluconate and optionally at least one zinc salt selected from the group consisting of zinc lactate, zinc picolinate and zinc tartrate.
 31. The composition according to claim 3 wherein said zinc salt is at least one zinc salt selected from the group consisting of zinc lactate, zinc picolinate and zinc tartrate and optionally at least one zinc salt selected from the group consisting of zinc gluconate, zinc acetate and zinc chloride.
 32. The composition according to claim 13 wherein said proton pump inhibitor is esomeprazole, lansoprazole, omeprazole, pantoprazole, rabeprazole or a mixture thereof.
 33. The composition according to claim 14 wherein said proton pump inhibitor is esomeprazole, lansoprazole, omeprazole, pantoprazole, rabeprazole or a mixture thereof.
 34. The composition according to claim 15 wherein said proton pump inhibitor is esomeprazole, lansoprazole, omeprazole, pantoprazole, rabeprazole or a mixture thereof.
 35. The composition according to claim 32 wherein said proton pump inhibitor is omeprazole.
 36. The composition according to claim 33 wherein said proton pump inhibitor is omeprazole.
 37. The composition according to claim 34 wherein said proton pump inhibitor is omeprazole. 